Advertisement

Organoids pp 43-53 | Cite as

Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres

  • Robert E. Hynds
  • Colin R. Butler
  • Sam M. Janes
  • Adam GiangrecoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1576)

Abstract

Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy.

Keywords:

Lung Stem cells Epithelial cells Goblet cells Cilia Adult stem cells Cell culture techniques Primary cell culture 

Notes

Acknowledgments

We thank Prof. Richard Schlegel (Georgetown University, USA), Xuefeng Liu (Georgetown University, USA), and Dr. Henry Danahay (University of Sussex, UK) for sharing their laboratories protocols during the development of those described here.

This work was supported by a BBSRC-CASE studentship with industrial support from Unilever (R.E.H.), a Wellcome Trust Clinical Research Fellowship (C.R.B.), an ERC Starting Grant (A.G.), a Wellcome Trust Senior Fellowship (S.M.J.) and was undertaken at UCLH/UCL who receive funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme and the UCL Experimental Cancer Medicine Centre (S.M.J.).

References

  1. 1.
    Hogan BL, Barkauskas CE, Chapman HA, Epstein JA, Jain R, Hsia CC, Niklason L, Calle E, Le A, Randell SH, Rock J, Snitow M, Krummel M, Stripp BR, Vu T, White ES, Whitsett JA, Morrisey EE (2014) Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15:123–138CrossRefGoogle Scholar
  2. 2.
    Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, Randell SH, Hogan BL (2009) Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 106:12771–12775CrossRefGoogle Scholar
  3. 3.
    Teixeira VH, Nadarajan P, Graham TA, Pipinikas CP, Brown JM, Falzon M, Nye E, Poulsom R, Lawrence D, Wright NA, McDonald S, Giangreco A, Simons BD, Janes SM (2013) Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors. Elife 2, e00966CrossRefGoogle Scholar
  4. 4.
    Hynds RE, Giangreco A (2013) Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine. Stem Cells 31:417–422CrossRefGoogle Scholar
  5. 5.
    de Jong PM, van Sterkenburg MA, Hesseling SC, Kempenaar JA, Mulder AA, Mommaas AM, Dijkman JH, Ponec M (1994) Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. Am J Respir Cell Mol Biol 10:271–277CrossRefGoogle Scholar
  6. 6.
    Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH (2005) Well-differentiated human airway epithelial cell cultures. Methods Mol Med 107:183–206PubMedGoogle Scholar
  7. 7.
    Mathis C, Poussin C, Weisensee D, Gebel S, Hengstermann A, Sewer A, Belcastro V, Xiang Y, Ansari S, Wagner S, Hoeng J, Peitsch MC (2013) Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. Am J Physiol Lung Cell Mol Physiol 304:L489–L503CrossRefGoogle Scholar
  8. 8.
    Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343CrossRefGoogle Scholar
  9. 9.
    Chapman S, Liu X, Meyers C, Schlegel R, McBride AA (2010) Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest 120:2619–2626CrossRefGoogle Scholar
  10. 10.
    Suprynowicz FA, Upadhyay G, Krawczyk E, Kramer SC, Hebert JD, Liu X, Yuan H, Cheluvaraju C, Clapp PW, Boucher RC Jr, Kamonjoh CM, Randell SH, Schlegel R (2012) Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells. Proc Natl Acad Sci U S A 109:20035–20040CrossRefGoogle Scholar
  11. 11.
    Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180:599–607CrossRefGoogle Scholar
  12. 12.
    Butler CR, Hynds RE, Gowers KH, Lee Ddo H, Brown JM, Crowley C, Teixeira VH, Smith CM, Urbani L, Hamilton NJ, Thakrar RM, Booth HL, Birchall MA, De Coppi P, Giangreco A, O'Callaghan C, Janes SM (2016) Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering. Am J Respir Crit Care Med194(2):156–168. doi: 10.1164/rccm.201507-1414OC, PubMed PMID: 26840431CrossRefGoogle Scholar
  13. 13.
    Wu X, Peters-Hall JR, Bose S, Pena MT, Rose MC (2011) Human bronchial epithelial cells differentiate to 3D glandular acini on basement membrane matrix. Am J Respir Cell Mol Biol 44:914–921CrossRefGoogle Scholar
  14. 14.
    Hegab AE, Ha VL, Darmawan DO, Gilbert JL, Ooi AT, Attiga YS, Bisht B, Nickerson DW, Gomperts BN (2012) Isolation and in vitro characterization of basal and submucosal gland duct stem/progenitor cells from human proximal airways. Stem Cells Transl Med 1:719–724CrossRefGoogle Scholar
  15. 15.
    Danahay H, Pessotti AD, Coote J, Montgomery BE, Xia D, Wilson A, Yang H, Wang Z, Bevan L, Thomas C, Petit S, London A, LeMotte P, Doelemeyer A, Velez-Reyes GL, Bernasconi P, Fryer CJ, Edwards M, Capodieci P, Chen A, Hild M, Jaffe AB (2015) Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung. Cell Rep 10:239–252CrossRefGoogle Scholar
  16. 16.
    Gao X, Bali AS, Randell SH, Hogan BL (2015) GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells. J Cell Biol 211:669–682CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Open Access This chapter is licensed under the terms of the Creative Commons Attribution-NonCommercial 2.5 International License (http://creativecommons.org/licenses/by-nc/2.5/), which permits any noncommercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Authors and Affiliations

  • Robert E. Hynds
    • 1
  • Colin R. Butler
    • 1
  • Sam M. Janes
    • 1
  • Adam Giangreco
    • 1
    Email author
  1. 1.Lungs for Living Research Centre, UCL RespiratoryUniversity College LondonLondonUK

Personalised recommendations